Tissue factor for influencing blood vessel formation

ABSTRACT

The present invention relates to the use of tissue factor for influencing blood vessel formation, particularly for activating blood vessel formation, above all for wound healing.

This is a national phase filing of the Application No. PCT/DE98/01306,which was filed with the Patent Corporation Treaty on May 8, 1998, andis entitled to priority of the and is a continuation of PCT/DE98/01278filed May 7, 1998.

FIELD OF THE INVENTION

The present invention relates to the use of issue factor for influencingblood vessel formation, particularly for activating blood vesselformation, above all for wound healing.

BACKGROUND OF THE INVENTION

The body is provided with blood by means of blood vessels. Blood vesselscomprise endothelial and smooth muscle cells. In many diseases, bloodvessels and the formation thereof, respectively, are impaired. This isfound, e.g., in impaired wound healing as in the case of diabetesmellitus, vasculitis, arterial occlusive disease, chronic venous andinfected ulcer. There are also major problems in connection with woundhealing in the case of innervation impairment such as paraplegia,leprosy, neuropathy, etc., and decubital gangrene of persons in need ofcare. Also known are weak sutures and wound healing impairment in thecase of operations, particularly of the intestines and transplantationsof skin or other organs, respectively. Up to the present, there are nosatisfactory products or means by which it is possible to take steps inthe case of blood vessel diseases, in particular impaired wound healing.

Therefore, it is the object of the present invention to provide aproduct by means of which the above objective can be achieved.

SUMMARY OF THE INVENTION

The present invention relates to the use of tissue factor forinfluencing blood vessel formation, particularly for activating bloodvessel formation, above all for wound healing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the formation of blood-vessels in wounds transfected with atissue factor-expressing vector. FIG. 1A shows the formation of bloodvessels in wounds tranfected with a tissue factor-expressing vector (a).FIG. 1B shows the formation of blood vessels in wounds transfected witha vector which codes for an antisense tissue factor (b). FIG. 1C showsthe control.

FIG. 2A shows stains using hemtoxylin/eosin staining to make the bloodvessels visible. FIG. 2A-1 shows the formation of vessels in woundstransfected with a tissue factor-expressing vector (a). FIG. 2A-2 showsthe formation of blood vessels in wounds transfected with a vector whichcodes for an antisense tissue factor (b). FIG. 2A-3 shows the control.

FIG. 2B shows the number of blood vessels by way of a diagram.

FIG. 3 shows the presence of smooth muscle cells in newly formed vesselsusing α-actin staining to make the vessels visible. FIG. 3A-1 shows theformation of vessels in wounds transfected with a tissuefactor-expressing vector (a). FIG. 3A-2 shows the formation of bloodvessels in wounds transfected with a vector which codes for an antisensetissue factor (b) FIG. 3A-3 shows the control.

FIG. 3B shows the strength of the staining by way of a diagram.

DETAILED DESCRIPTION OF THE INVENTION

It is the object of the present invention to provide a product by meansof which the above objective can be achieved. According to the inventionthis is achieved by the subject matters defined in the claims.

Thus, the subject matter of the present invention relates to the use oftissue factor for influencing blood vessel formation, in particular foractivating blood vessel formation, above all for wound healing.

The present invention is based on the applicant's finding that in woundsof animals tissue factor results in the formation of vessels (bloodvessels). He found out that the vessels comprise endothelial and smoothmuscle cells. The applicant also recognized that wound healing can beachieved by means of tissue factor. Furthermore, the applicantdiscovered that vessel formation can be prevented by inhibiting tissuefactor.

Tissue factor is a transmembrane glycoprotein which binds the bloodclotting factors VII and VITa, respectively. An activation of the bloodclotting factors X and IX, respectively, is effected by this bond, sothat the blood coagulation is started via the extrinsic path andintrinsic path, respectively. Tissue factor has a molecular weight of 43to 46 kD. Its primary structure is known as is the gene for tissuefactor and its localization on the chromosome. Scarpati et al, 1987,Biochemistry 26:5234-5238.

According to the invention tissue factor is used for activating vesselformation, particularly for wound healing. The expression “tissuefactor” relates to a tissue factor of any kind and origin. It may be ananimal or human tissue factor. It can be glycosylated ornon-glycosylated. Also, it may be a fragment of tissue factor which iscapable of forming vessels, in particular for wound healing. The tissuefactor can have a wild-type sequence. Its sequence can also differ fromthe wild-type sequence by one or several amino acids. In addition, thetissue factor can be part of a fusion protein.

In a preferred embodiment, the tissue factor is present in the form ofan expressible nucleic acid. It may be a DNA and/or RNA, a DNA,particularly a genomic or cDNA and fragments thereof, respectively,being preferred. The above statements made on the tissue factor applyhere correspondingly to the nucleic acid.

The expression of the nucleic acid can be achieved as usual. It can befavorable for the nucleic acid, e.g., as a DNA, particularly cDNA, to bepresent in a vector which is suitable for expression in animal cells. Aperson skilled in the art is familiar with such expression vectors. Forexample, they may be virus or plasmid vectors. It is advantageous forthe vectors not to integrate into the genome of cells but remainepisomally within the cells. By this, a transient expression of thetissue factor is achieved, which is preferred. The nucleic acid as aDNA, particularly cDNA, can also be controlled by a constitutive orinducible promoter. An inducible promoter can be, e.g., tissue-, organ-and/or tumor-specific. It can be favorable for the nucleic acid as DNA,particularly cDNA, to be controlled by the CMV promoter, e.g., in theexpression vector pcDNA3 (Invitrogen company) or controlled by the SV40promoter, e.g., in the expression vector pSVK3 (Pharmacia company). Suchexpression plasmids referred to as pcDNA3-TF (tissue factor) andpSVK3-TF, respectively, also represent a subject matter of the presentinvention. It can be particularly advantageous for the nucleic acids asDNA, particularly cDNA, to be present in a Sindbis virus repliconvector. Such a vector permits an extremely high expression of thenucleic acid. An example of such a vector is the ELVS vector system fromViagene Inc. An expression plasmid referred to as ELVS-TF (tissuefactor) also represents a subject matter of the present invention. Forthe preparation of an above vector, a person skilled in the art will useknown methods. Reference is made to Maniatis et al., 1982, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, by way ofsupplement.

According to the invention tissue factor is used for activating vesselformation, in particular for wound healing. The expression “vesselformation” relates to a vessel formation of any kind and at any site.For example, it relates to a vessel formation serving for replacingimpaired,. e g., old, blood vessels. They can be present, e.g., in thebrain or heart, so that an apoplexy or infarction can be prevented ortreated. Precautions can also be taken against presbyphrenia. Inaddition, it relates to a vessel formation for treatingarteriosclerosis, Crohn's disease and ulcerative colitis, diabeticretinopathy and deep venous thrombosis of the legs/ulcus cruris as wellas the prevention of relapses. In particular, it is relates to vesselformation and wound healing. The expression “wound healing” relates towound healing of any kind and at any site. It can be normal and impairedwound healing. The latter is found in particular in the case ofdiseases, such as diabetes mellitus, vasculitis, arterial occlusivedisease, chronic venous and/or infected ulcer as well as poorly healinggastric ulcer. Impaired wound healing is also found in the case ofinnervation impairment such as paraplegia, leprosy, neuropathy, etc.,and decubital gangrene of persons in need of care. Impaired woundhealing will also be given if weak sutures and impaired healing occurafter operations, particularly of the intestines and transplantations ofskin and other organs, respectively. Impaired wound healing is alsofound in the case of bone fractures, burns and treatments usingsteroids.

According to the invention tissue factor is administered in the form ofa protein or an expressible nucleic acid to activate vessel formation,in particular for wound healing. It may be favorable for the tissuefactor to be administered in combination with further factors supportingvessel formation, in particular for wound healing, such as vascularendothelial growth factor (VEGF). These factors can also be present inthe form of proteins and/or expressible nucleic acids. The tissue factorand said factors can be administered simultaneously or successively. Thekind of administration of tissue factor alone and together with saidfactors, respectively, can orient itself by the site of action, i.e., atthe site where blood vessel formation, in particular for wound healing,shall take place. For example, it is an obvious thing to treat an areaon the body surface locally and one within the interior of the bodysystemically. Common methods can be used for the administration oftissue factor alone and together with said factors, respectively. Forthe local administration it is, e.g., favorable to pack the factor orfactors into liposomes or absorb them onto carriers, particularly goldparticles, and apply the liposomes to the corresponding site of the bodyand shoot the carriers, particularly gold particles, into the tissue,respectively. Furthermore, pharmaceutical compositions are provided forthe administration of tissue factor alone and together with saidfactors, respectively, which may contain common auxiliary substancessuch as carriers, solvents, etc. Such compositions also represent asubject mater of the present invention.

According to the invention tissue factor is also used for inhibitingblood vessel formation. For this purpose, the tissue factor can bepresent in the form of an antibody inhibiting it. The tissue factor canalso be present in the form of a nucleic acid which has an antisenseeffect on the expression of tissue factor. In particular tumoraldiseases can be treated by the inhibition of vessel formation.

By means of the present invention it is possible to influence vesselformation. In particular, vessel formation can be activated. Theresulting blood vessels comprise endothelial and smooth muscle cells.Thus, the present invention is suited for the prevention and treatmentof the most varying diseases. Examples thereof are indicated above. Inparticular, the present invention is suitable for the treatment and/orprophylaxis of impaired wound healing, above all in the case of diabetesmellitus, where it is possible to heal large open wounds located at theextremities. In addition, vessel formation can be inhibited by means ofthe present invention. Thus, the present invention is also suited totreat diseases, such as tumoral diseases. The present invention makes amajor contribution to modern medicine.

The below examples explain the invention in more detail. The followingpreparations and examples are given to enable those skilled in the artto more clearly understand and to practice the present invention. Thepresent invention, however, is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only, and methods which are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

EXAMPLE

Preparation of a Tissue Factor-expressing Plasmid and its use forInfluencing Blood Vessel Formation, Particularly for Activating BloodVessel Formation, Above All for Wound Healing

(A) The entire translated region (1.8 kb) of the mouse tissue factorgene was integrated into the BamHI site of the multiple cloning site ofpcDNA3 (Invitrogen). Thus, this region was controlled by the CMVpromoter. The expression plasmid pcDNA3-TF was obtained. In the sameway, the coding region (0.7 kb) of the mouse tissue factor gene wasintegrated in the antisense orientation into the EcoRI site of themultiple cloning site of pcDNA3. Thus, this region was also controlledby the CMV promoter. The expression plasmid pcDNA3-TF-AS was obtained.

6 mm full thickness wounds each were placed on the backs of three femaleNOD mice (Bomholtgaard, Denmark) at a distance of 8 to 10 mm. Thesewounds were treated with mixtures containing 2 μg pcDNA3-TF (a),pcDNA3-TF-AS (b) and pcDNA3 (control (c)), respectively, and 12 μg DOTAPtransfection reagent (Boehringer Mannheim) each. The wounds were coveredwith Ohmann Opraflex.

For proving the formation of vessels (blood vessels) in the wounds, 300μl of ink (Nigrosin, Sigma) each were injected into the caudal vein ofthe mice 6 days and 8 days, respectively, following the administrationof the mixtures. Thereafter, the animals were killed and the skinregions with the wounds were examined under a microscope.

It showed that if a tissue factor-expressing vector (a) is administered,vessels (blood vessels) will be formed in wounds and thus wound healingwill be promoted. It also turned out that an antisense tissue factor caninhibit the formation of blood vessels.

(B) As described under (A), six NOD mice were treated. After 6 days and8 days, respectively, the animals were killed and the corresponding skinregions were examined under a microscope after having been subjected toα-actin staining (with Sm-actin antibodies from Dianova).

It showed that the blood vessels formed comprise smooth muscle cells.

All references cited within the body of the instant specification arehereby incorporated by reference in their entirety.

1. A method of activating blood vessel formation in a subject in need,comprising locally administering a functional Tissue Factor in atherapeutically effective amount to a non-malignant tissue of saidsubject in need, wherein said Tissue Factor or a fragment thereof isadministered in the form of an expressible nucleic acid present in aplasmid vector; and wherein the administration of the functional TissueFactor causes activation of blood vessel formation in the non-malignanttissue.
 2. The method of claim 1, wherein said nucleic acid is expressedtransiently.
 3. The method of claim 1, wherein said nucleic acid is aDNA.
 4. The method of claim 1, wherein said nucleic acid is controlledby a constitutive or an inducible promoter.
 5. The method of claim 1,wherein said nucleic acid is controlled by a CMV or SV40 promoter. 6.The method of claim 1, wherein the said subject in need is afflictedwith diabetes mellitus, vasculitis, arterial conclusive disease, chronicvenous and infected ulcer, innervation impairment, decubital gangrene orweak sutures after a surgery.
 7. The method of claim 1, wherein saidsubject in need is afflicted with arteriosclerosis, Chroh's disease,ulcerative colitis, diabetic retinopathy, or deep venous thrombosis ofthe legs ulcus cruris.
 8. The method of claim 1, wherein the bloodvessel formation is activated for the replacement of impaired bloodvessels.
 9. A method for enhancing wound healing in a subject in need,comprising locally administering a Tissue Factor or a fragment thereofin the form of an expressible nucleic acid present in a plasmid vectorto a non-malignant wound tissue of said subject in need.
 10. The methodof claim 1 or 9, wherein said plasmid vector is present in a liposome oron a carrier.
 11. The method of claim 10, wherein said carrier is a goldparticle.
 12. The method of claim 1 or 9, wherein said plasmid vector ispresent in a pharmaceutical composition.
 13. The method of claim 12,wherein said plasmid vector is present in combination with furtherfactors promoting the formation of blood vessels.
 14. The method ofclaim 13, wherein said further factors are present as expressiblenucleic acids or functional proteins.
 15. The method of claim 14,wherein at least one further factor is present and said further factoris VEGF.